Here we report the discovery of two Tupanvirus strains, the longest tailed Mimiviridae members isolated in amoebae. Their genomes are 1.44-1.51 Mb linear double-strand DNA coding for 1276-1425 predicted proteins. Tupanviruses share the same ancestors with mimivirus lineages and these giant viruses present the largest translational apparatus within the known virosphere, with up to 70 tRNA, 20 aaRS, 11 factors for all translation steps, and factors related to tRNA/mRNA maturation and ribosome protein modification. Moreover, two sequences with significant similarity to intronic regions of 18 S rRNA genes are encoded by the tupanviruses and highly expressed. In this translation-associated gene set, only the ribosome is lacking. At high multiplicity of infections, tupanvirus is also cytotoxic and causes a severe shutdown of ribosomal RNA and a progressive degradation of the nucleus in host and non-host cells. The analysis of tupanviruses constitutes a new step toward understanding the evolution of giant viruses.
Abstract The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4−/− mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which was prevented in TLR4−/− macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4−/− macrophages, suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages. These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii were similar in wild-type and TLR4−/− mice. We also showed that the number of granulomas was decreased in the liver of infected TLR4−/− mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associated with decreased production of IFN-γ and TNF. Taken together, these results demonstrate that TLR4 controls early events of C. burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.
Taxonogenomics combines phenotypic assays and genomic analysis as a means of characterizing novel strains. We used this strategy to study Marseille-P9898T strain, an aerobic, motile, Gram-negative, spore-forming, and rod-shaped bacterium isolated from planarian Schmidtea mediterranea. Marseille-P9898T is catalase-positive and oxidase-negative. The major fatty acids detected are 12-methyl-tetradecanoic acid, 13-methyl-tetradecanoic acid, and hexadecanoic acid. Marseille-P9898T strain shared more than 98% sequence similarity with the Metabacillus niabensis strain 4T19T (98.99%), Metabacillus halosaccharovorans strain E33T (98.75%), Metabacillus malikii strain NCCP-662T (98.19%), and Metabacillus litoralis strain SW-211T (97.15%). Marseille-P9898 strain belongs to Metabacillus genus. Genomic analysis revealed the highest similarities with Ortho-ANI and dDDH, 85.76% with Metabacillus halosaccharovorans, and 34.20% with Bacillus acidicola, respectively. These results show that the Marseille-P9898T strain is a novel bacterial species from Metabacillus genus, for which we propose the name of Metabacillus schmidteae sp. nov. (Type strain Marseille-P9898T = CSUR P9898T = DSM 111480T).
Viruses must enter host cells to deliver their genetic material and accessory proteins. Endocytosis offers to viruses the opportunity to enter host cells. However, endocytosis is a complex phenomenon that includes different mechanisms, clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, and phagocytosis. Here, I describe the ways used by different viruses to exploit these endocytic pathways.
Dendritic cells (DCs) orchestrate host defenses against microorganisms. In infectious diseases due to intracellular bacteria, the inefficiency of the immune system to eradicate microorganisms has been attributed to the hijacking of DC functions. In this study, we selected intracellular bacterial pathogens with distinct lifestyles and explored the responses of monocyte-derived DCs (moDCs). Using lipopolysaccharide as a control, we found that Orientia tsutsugamushi, the causative agent of scrub typhus that survives in the cytosol of target cells, induced moDC maturation, as assessed by decreased endocytosis activity, the ability to induce lymphocyte proliferation and the membrane expression of phenotypic markers. In contrast, Coxiella burnetii, the agent of Q fever, and Brucella abortus, the agent of brucellosis, both of which reside in vacuolar compartments, only partly induced the maturation of moDCs, as demonstrated by a phenotypic analysis. To analyze the mechanisms used by C. burnetii and B. abortus to alter moDC activation, we performed microarray and found that C. burnetii and B. abortus induced a specific signature consisting of TLR4, TLR3, STAT1 and interferon response genes. These genes were down-modulated in response to C. burnetii and B. abortus but up-modulated in moDCs activated by lipopolysaccharide and O. tsutsugamushi. This transcriptional alteration was associated with the defective interferon-β production. This study demonstrates that intracellular bacteria specifically affect moDC responses and emphasizes how C. burnetii and B. abortus interfere with moDC activation and the antimicrobial immune response. We believe that comparing infection by several bacterial species may be useful for defining new pathways and biomarkers and for developing new treatment strategies.
Macrophages are the first line of defense against pathogens. Upon infection macrophages usually produce high levels of proinflammatory mediators. However, macrophages can undergo an alternate polarization leading to a permissive state. In assessing global macrophage responses to the bacterial agent of Whipple's disease, Tropheryma whipplei, we found that T. whipplei induced M2 macrophage polarization which was compatible with bacterial replication. Surprisingly, this M2 polarization of infected macrophages was associated with apoptosis induction and a functional type I interferon (IFN) response, through IRF3 activation and STAT1 phosphorylation. Using macrophages from mice deficient for the type I IFN receptor, we found that this type I IFN response was required for T. whipplei-induced macrophage apoptosis in a JNK-dependent manner and was associated with the intracellular replication of T. whipplei independently of JNK. This study underscores the role of macrophage polarization in host responses and highlights the detrimental role of type I IFN during T. whipplei infection.
Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus, increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus (M. abscessus) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus, testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus, we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a, necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near-complete absence of GPL on the bacterial surface is a necessary condition for optimal pathogenesis of this mycobacterium. They also suggest that GPL content affects hydrophobicity of M. abscessus, potentially altering the aerosol transmission, which is of particular importance from an epidemiological and clinical perspective.
Abstract Despite increasing evidence indicates polyamines as a convergence point for signaling pathways, including cell growth and differentiation, a unifying concept to interpret their role is still missing. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is tightly regulated by a complex molecular machinery, and the demonstration of the existence of multiple ODC paralogs, lacking decarboxylation activity, suggests additional layers of complexity to the intricate ODC regulatory pathway. Because of their extraordinary regenerative abilities and abundance of stem cells, planarians have potential to contribute to our understanding of polyamine function in an in vivo context. We undertook a study on ODC function in planarians and we found six planarian ODCs ( ODC1-6 ). Five out of six ODC homologs carry substitutions of key aminoacids for enzymatic activity, which makes them theoretically unable to decarboxylate ornithine. Silencing of ODC5 and 6 produced a complex phenotype, by prompting animals to an aberrant response, following chronic injury without tissue removal. Phenotype is neither rescued by putrescine, nor mimicked by difluoromethylornithine treatment. Moreover, the co-silencing of other genes of the ODC regulatory pathway did not modulate phenotype outcome or severity, thus suggesting that the function/s of these ODC-like proteins might be unrelated to decarboxylase activity and putrescine production.
SUMMARY As Q fever is associated with an inflammatory syndrome, we determined circulating levels of inflammatory cytokines, cytokine antagonists, and activation markers of leucocytes in patients with acute Q fever and Q fever endocarditis. Tumour necrosis factor (TNF) and IL-6, but not IL-1β, were markedly increased compared with controls. Cytokine antagonists and activation markers of leucocytes were profoundly different in acute and chronic Q fever. IL-1 receptor antagonist and TNF receptor type II were significantly increased in patients with acute Q fever, suggesting a shift of cytokine balance towards cytokine antagonists. The activation marker of B cells, sCD23, was significantly increased in Q fever endocarditis compared with controls and patients with acute Q fever. In a 2-year follow-up study of patients with Q fever endocarditis, sCD23 and specific IgG levels slowly decreased in patients whose symptoms resolved, but remained high in those who required prolonged treatment.
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